Reactive oxygen species are known to damage various biomolecules, including lipids, proteins, and nucleic acid. But there is growing evidence that reactive oxygen species can also serve as second messengers in cell signaling pathways that regulate cell proliferation and cell death (apoptosis). Because hydrogen peroxide is one of the most physiologically important reactive oxygen species, we have investigated its role in cellular toxicity and in cell signaling in HeLa cells. Hydrogen peroxide was administered by two different methods: (1) by direct addition of a hydrogen peroxide solution and (2) by addition of glucose and glucose oxidase to generate low levels of hydrogen peroxide continuously. Both treatments resulted in cytotoxicity, decreased catalase activity, and depletion of cellular thiol levels. However, the glucose/glucose oxidase system resulted in inactivation of caspase 3 activity, which was restored after removal of the system. To the contrary, caspase 3 activity was elevated rapidly following addition of a bolus of hydrogen peroxide and remained elevated for 24 hours thereafter. In neither case, however, was DNA condensation (a measure of apoptosis) observed by means of fluorescence microscopy. However, treatment with hydrogen peroxide led to phosphorylation of various mitogen activated protein kinases (e.g. P38, JNK, and ERK) that are often involved in apoptosis. Direct addition of hydrogen peroxide led also to a slight decrease in telomerase and protein kinase B (Akt) levels as evidenced by Western blot analysis. However, phosphorylation of Akt was elevated by the hydrogen peroxide treatment. Since phosphorylation of Akt is involved in both anti- apoptosis and stimulation of telomerase activity, these results suggest that Akt may be a major target for hydrogen peroxide-dependent regulation of cell proliferation and/or apoptosis pathways. Future experiments will focus on the upstream and downstream targets of Akt in hydrogen peroxide treated HeLa cells, and delineation of their roles in the regulation of cellular activities. - reactive oxygen species, caspase 3, MAPK, Akt, apoptosis